Limestone aquifers in Southern Italy are often affected by bacterial contamination produced by pasture and agriculture. The main goals of this study were (1) to analyze the role of land use and environmental factors on microbial contamination and, (2) to identify, at field scale, the most suitable indicator of fecal pollution, by comparing fecal coliforms and fecal enterococci. Analyzing surface and spring water, it was noted that both fecal indicators showed a significant decrease during the period characterized by freezing and/or freeze-thaw intervals. The data analysis shows that fecal coliforms are characterized by a significant decrease in population (3 orders of magnitude, at least) during the freezing period, while fecal enterococci are temporarily inhibited. A taxonomic classification of fecal enterococci detected in spring water samples was performed by the API 20 Strep system and by sequencing of the ribosomal 16S DNA genes. The results showed that freezing conditions did not cause any significant change on the set of enterococcal species.